Method for generating diversity
View Patent ↗The invention relates to a method for preparing an antibody-producing cell line capable of directed constitutive hypermutation of a specific nucleic acid region, comprising the steps of: a) screening a clonal cell population for V gene diversity; b) isolating one or more cells which display V gene diversity and comparing the rate of accumulation of mutations in the V genes and other genes of the selected cells; and c) selecting a cell in which the rate of V gene mutation exceeds that of other gene mutation.
1. A method for preparing a B cell line capable of directed constitutive hypermutation of a target nucleic acid region, the method comprising: screening a B cell population for ongoing target sequence diversification, wherein said screening comprises determining the mutation rate of the target nucleic acid region relative to the mutation rate of a non-target nucleic acid region, and selecting a B cell from said population in which the mutation rate of the target nucleic acid region in the selected B cell exceeds that of the non-target nucleic acid region in the selected B cell by a factor of 100 or more, whereby a B cell line exhibiting directed constitutive hypermutation of the target nucleic acid region is prepared.
2. A method of claim 1 , wherein the B cell is a B cell lymphoma cell.
3. A method of claim 2 , wherein the B cell is one selected from the group consisting of a Burkitt lymphoma cell, a follicular lymphoma cell, a Ramos cell and diffuse large B cell lymphoma cell.
4. A method of claim 1 , wherein the target nucleic acid region comprises an immunoglobulin V-gene sequence.
5. A method of claim 1 , wherein the non-target nucleic acid region comprises an immunoblogulin C-gene sequence.
6. A method of claim 1 , wherein the mutation rate is determined by sequencing the target nucleic acid region and the non-target nucleic acid region.
7. A method of claim 1 , wherein the mutation rate is determined by a non-sequencing method selected from the group consisting of a Muts assay, an assay assessing IgM loss, and an assay assessing a change in binding affinity.